The underlying progression of genetic events which transform a normal cell into a cancer cell is characterized by a shift from the diploid to anueploid state (Albertson et al. (2003), Nat Genet, Vol. 34, pp. 369-76 and Lengauer et al. (1998), Nature, Vol. 396, pp. 643-9). As a result of genomic instability, cancer cells accumulate both random and causal alterations at multiple levels from point mutations to whole-chromosome aberrations. DNA copy number changes include, but are not limited to, loss of heterozygosity (LOH) and homozygous deletions, which can result in the loss of tumor suppressor genes, and gene amplification events, which can result in cellular proto-oncogene activation. One of the continuing challenges to unraveling the complex karyotype of the tumor cell is the development of improved molecular methods that can globally catalogue LOH, gains, and losses with both high resolution and accuracy.
Numerous molecular approaches have been described to identify genome-wide LOH and copy number changes within tumors. Classical LOH studies designed to identify allelic loss using paired tumor and blood samples have made use of restriction fragment length polymorphisms (RFLP) and, more often, highly polymorphic microsatellite markers (STRS, VNTRs). The demonstration of Knudson's two-hit tumorigenesis model using LOH analysis of the retinoblastoma gene, Rb1, showed that the mutant allele copy number can vary from one to three copies as the result of biologically distinct second-hit mechanisms (Cavenee, et al. (1983), Nature, Vol. 305, pp. 779-84.). See also Knudson, A. G., Cancer Res. 45, 1437-1443 (1985). Thus regions undergoing LOH do not necessarily contain DNA copy number changes.
Approaches to measure genome wide increases or decreases in DNA copy number include comparative genomic hybridization (CGH) (Kallioniemi, et al. (1992), Science, Vol. 258, pp. 818-21.), spectral karyotyping (SKY) (Schrock, et al. (1996), Science, Vol. 273, pp. 494-7.), fluorescence in situ hybridization (FISH) (Pinkel et al. (1988), Proc Natl Acad Sci USA, Vol. 85, pp. 9138-42), molecular subtraction methods, such as RDA (Lisitsyn et al. (1995), Proc Natl Acad Sci USA, Vol. 92, pp. 151-5.; Lucito et al. (1998), Proc Natl Acad Sci USA, Vol. 95, pp. 4487-92), and digital karyotyping (Wang, et al. (2002), Proc Natl Acad Sci USA, Vol. 99, pp. 16156-61.). CGH, perhaps the most widely used approach, uses a mixture of DNA from normal and tumor cells that have been differentially labeled with fluorescent dyes. Target DNA is competitively hybridized to metaphase chromosomes or, in array CGH, to cDNA clones (Pollack et al. (2002), Proc Natl Acad Sci USA, Vol. 99, pp. 12963-8) or bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs) (Snijders et al. (2001), Nat Genet, Vol. 29, pp. 263-4, Pinkel, et al. (1998), Nat Genet, Vol. 20, pp. 207-11). See also, Balmain et al. Nature Genet. 33:238 (2003) and Fridlyand et al BMC Cancer 6:96 (2006). Hybridization to metaphase chromosomes, however, limits the resolution to 10-20 Mb, precluding the detection of small gains and losses. While the use of arrayed cDNA clones allows analysis of transcriptionally active regions of the genome, the hybridization kinetics may not be as uniform as when using large genomic clones. Currently, the availability of BAC clones spanning the genome limits the resolution of CGH to 1-2 Mb. CGH, however, is not well-suited to identify regions of the genome which have undergone LOH such that a single allele is present but there is no reduction in copy number.
With the completion of the human genome, single nucleotide polymorphisms (SNPs), the most common sequence variation among individuals, are emerging as the marker of choice in large-scale genetic studies due to their abundance, stability, and relative ease of scoring. These same characteristics make SNPs powerful markers for LOH studies.